Analysis of clinical phenotypic and genotypic spectra in 36 children patients with Epilepsy of Infancy with Migrating Focal Seizures

Epilepsy of Infancy with Migrating Focal Seizures (EIMFS) is a rare developmental and epileptic encephalopathy (DEEs) with unknown etiology, and poor prognosis. In order to explore new genetic etiology of EIMFS and new precision medicine treatment strategies, 36 children with EIMFS were enrolled in this study. 17/36 cases had causative variants across 11 genes, including 6 novel EIMFS genes: PCDH19, ALDH7A1, DOCK6, PRRT2, ALG1 and ATP7A. 13/36 patients had ineffective seizure control, 14/36 patients had severe retardation and 6/36 patients died. Of them, the genes for ineffective seizure control, severe retardation or death include KCNT1, SCN2A, SCN1A, ALG1, ATP7A and WWOX. 17 patients had abnormal MRI, of which 8 had ineffective seizure control, 7 had severe retardation and 4 died. 13 patients had hypsarrhythmia, of which 6 had ineffective seizure control, 6 had severe retardation and 2 died. Also, 7 patients had burst suppression, of which 1 had ineffective seizure control, 3 had severe retardation and 3 died. This study is the first to report that ALDH7A1, ATP7A, DOCK6, PRRT2, ALG1, and PCDH19 mutations cause the phenotypic spectrum of EIMFS to expand the genotypic spectrum. The genes KCNT1, SCN2A, SCN1A, ALG1, ATP7A and WWOX may be associated with poor prognosis. The patients presenting with MRI abnormalities, hypsarrhythmia and burst suppression in EEG may be associated with poor prognosis.

KCNT1 EIMFS patients (n = 3). Three patients (3 males) with KCNT1 variants aged 1 year and 4 months to 2 years were studied (median 1 year 4months). The median seizure onset was 1 months (range 15 days to 4 months). All had focal seizures, 1 had tonic seizures, 2 had spasms seizures, and 2 had autonomic seizures. All had normal brain imaging findings. EEG showed seizure migration in all patients, hypsarrhythmia in 2/3, and burst suppression in 1/3. Two patients had severe developmental retardation, and one died. The age of death was 2 years. www.nature.com/scientificreports/ SCN2A EIMFS (n=6). There were 6 patients with SCN2A EIMFS studied at 1 month to 6 years of age (median 1 years 5 months) with median seizure onset at 2 days (range 1 day to 9 months). All had focal seizures, 4 had tonic seizures,2 had spasms seizures, and 1 had an autonomic seizure. A total of 2/6 had normal brain imaging findings, and 4/6 had abnormal imaging findings. EEG data showed seizure migration in all patients, hypsar- www.nature.com/scientificreports/ rhythmia in 3/6, and burst suppression in 2/6. The effective drugs for 6 patients with seizure control were oxcarbazepine, carbamazepine, vigabatrin and levetiracetam. Three patients had severe developmental retardation, three patients had mild-moderate development retardation, and no patients died.
PNPO and ALDH7A1 EIMFS (n=2). One patient (female) with PNPO variants was studied at 4 years and 4 months of age. Seizure onset was 2 days after birth. She had focal seizures and abnormal brain imaging findings. One patient (male) with ALDH7A1 variants was studied at 3 years and 10 months of age. Seizure onset was 1 month. He had focal and clonic seizures and normal brain imaging. There was no hypsarrhythmia or burst suppression in the EEG of these two patients. Vitamin B6 was effective in both patients. One patient had only mild development retardation, and one patient had normal development.
PRRT2 EIMFS (n=1). One patient (female) with PRRT2 variants was studied at 10.5 months of age. Seizure onset was 3 months. She had migarating focal seizures and normal brain imaging findings. Migarating focal seizures and no hypsarrhythmia or burst suppression was evident in the EEG data of the patient (Figure 1). Oxcarbazepine was effective in controlling seizures in this patient, who had normal development.

Treatment.
Anti seizure medication effects on seizures were analyzed in 36 patients with EIMFS (Table 3).
In addition to classical anti seizure medication, vitamin B6 and adrenocorticotropic hormones (ACTH) were used to treat EIMFS patients. Vitamin B6 allowed patients with ALDH7A1 and PNPO mutations to achieve seizure-free status, oxcarbazepine was effective for patients with SCN2A, ATP7A, WWOX, and PRRT2 mutations, and ACTH was partly effective for DOCK6 mutation patients with spasms and hypsarrhythmia.
We discovered 6 novel EIMFS genes in our cohort -de novo PCDH19; paternal ALDH7A1, DOCK6, PRRT2 and ALG1; and maternal ALDH7A1, ATP7A, DOCK6 and ALG1 -encoding a wide range of proteins. These genes have not been described in patients with EIMFS, but they have been associated with other neurological diseases. Considering that one of our patients was a female and had a de novo mutation of the PCDH19 gene, which was consistent with the X-linked genetic classification and the common clinical phenotype of spasms seizure and delayed motor development 12 , the software predicted the likely pathogenicity of the mutation; thus, these findings suggested that the PCDH19 gene could be associated with EIMFS. ALDH7A1 gene is associated with autosomal recessive pyridoxine-dependent epilepsy 13 . Our patient's clinical phenotype was recurrent epileptic seizures, accompanied by developmental delay, and vitamin B6 treatment was effective. The inheritance pattern was consistent with family co-segregation, and the software predicted it as likely pathogenic. Therefore, it can be considered as the causative gene of EIMFS. In future studies, it is hoped that more case reports, or functional studies, can confirm the pathogenicity of this variant. DOCK6 gene is associated with autosomal recessive Adams-Oliver syndrome-2, which is characterized by aplasia cutis congenita (ACC) as well as terminal transverse limb defects (TTLD) in addition to variable involvement of the brain, eyes, and cardiovascular system 14 . Our patient had developmental delays and recurrent seizures, which may be related to brain development disorders. Therefore, we could not determine whether the DOCK6 gene was the causative gene of EIMFS, and further data are needed for verification. It has been previously thought that PRRT2-associated paroxysmal movement disorders (PRRT2-PxMD) are mostly manifested as infantile-onset epilepsy and post-adolescent movement disorders, and most of them have a good prognosis and do not affect cognitive development. In addition, PRRT2 pathogenic variants have been identified in other childhood-onset movement disorders and different types of seizures, suggesting that the understanding of the spectrum of PRRT2-PxMD is still evolving 15 . The patient with this mutation in our www.nature.com/scientificreports/ cohort was very interesting. He presented with very frequent cluster seizures when he was 3 months old, video-EEG demonstrated migrating focal seizures and typical background characteristics of EIMFS, and he experienced obvious cognitive retardation after the onset of epilepsy. He achieved seizure-free status after the administration of oxcarbazepine, and follow-up showed normal cognitive development. The patient had a paternal mutation of the PRRT2 gene-his father had symptoms of dystonia in his youth-and the software predicted the likely pathogenicity of the mutation. We considered that the PRRT2 gene was the causative gene of EIMFS, suggesting that PRRT-2 spectrum diseases could also manifest as a very severe epileptic encephalopathy phenotype, but after effective treatment, the disease process could be quickly reversed and the prognosis was good. In the 10th patient, two genetic variants were detected simultaneously, namely, WWOX and ATP7A. The clinical phenotypes of WWOX gene reported in the literature were autosomal recessive epileptic encephalopathy, early infantile 28, esophageal squamous cell carcinoma, somatic and spinocerebellar ataxia, and autosomal recessive 12 16,17 . The WWOX gene was EIMFS-related genes reported in previous literature, which theoretically can cause epileptic encephalopathy. The clinical phenotypes of ATP7A gene reported in the literature were X-linked Menkes disease, occipital horn syndrome, distal spinal muscular atrophy 3 [18][19][20] . This patient also had a significant decrease in blue copper protein, had hair and skin color changes and could have been diagnosed with Menkes disease. At present, the epileptic seizure forms of Menke's disease reported in the literature include focal clonic seizures, myoclonic, and tonic seizures, infantile spasms, and no migrating focal seizures have been reported 21 . We could not confirm whether it was a copathogenic gene. ALG1 gene is associated with autosomal recessive congenital disorder of glycosylation type Ik 22,23 , which manifested as feeding problems and diarrhea, muscular hypertonia, refractory seizures, recurrent episodes of apnea, cardiac and hepatic involvement and coagulation anomalies 24 .
The clinical characteristics of this patient were consistent with the above core symptoms, the genetic results were consistent with the law of autosomal recessive inheritance, and the software predicted the likely pathogenicity of the mutation. We considered the ALG1 gene to be a newly pathogenic gene of EIMFS.
Most patients with EIMFS are refractory to anti-seizure medication, but some have shown good progression or a near satisfactory response to treatment. The anti-seizure medication used alone or in combination with one another that may achieve seizure control or reduction are potassium bromide, levetiracetam, ACTH, stiripentol, clonazepam, and rufinamide 25 . Mikati et al. reported that quinidine is effective in the treatment of patients with KCNT1 gene mutations in EIMFS 26 . Some studies have found that a correlation between age at disease onset, response to sodium channel blockers and the functional properties of mutations in children with SCN2A-related epilepsy. The use of sodium channel blockers was often associated with clinically relevant seizure reduction or seizure freedom in children with early infantile epilepsies (<3 months), whereas other antiepileptic drugs were less effective. In contrast, sodium channel blockers were rarely effective in epilepsies with later onset (≥3 months) and sometimes induced seizure worsening 27 . At present, there are few literature reports on the use of corresponding effective drug treatments for patients with different EIMFS gene mutations. In our cohort, we determined that vitamin B6 could allow patients with ALDH7A1 and PNPO mutations to achieve seizure-free status. Oxcarbazepine was effective for patients with SCN2A, ATP7A+WWOX, and PRRT2 mutations. Among our 6 children with SCN2A gene mutations, 4 of them were onset before 3 months of age, and the seizure control effect was good, which was consistent with the data reported in the literature. ACTH was partly effective for patients with DOCK6 mutations who had spasms and hypsarrhythmia.
While seizure outcomes and developmental prognoses are generally poor in EIMFS, there are rare reports of mildly affected patients 28 . In our study cohort, the incidence of poor prognosis was also relatively high; 6/36 (16.7%) patients died, and the related pathogenic genes were KCNT1, SCN1A, ALG1. 14/36 (38.9%) patients had severe retardation, and the genes for ineffective seizure control and severe retardation included KCNT1, SCN2A, WWOX and ATP7A. The results indicated that the related pathogenic genes KCNT1, SCN1A, ALG1, SCN2A, WWOX and ATP7A may be associated with ineffective seizure control and poor prognoses. While all patients experienced refractory epilepsy early in the course of the disease, 3/36 (8.3%) patients had normal mental and motor development. Genes associated with seizure-free, mild-moderate retardation or normal of mental and motor development included PRRT2, SCN2A, ALDH7A1, PCDH19 and PNPO.
In addition, we compared the association of MRI abnormalities, hypsarrhythmia and burst suppression in EEG with poor prognosis. The results found that patients with EIMFS characterized by abnormal MRI, hypsarrhythmia and burst suppression in EEG have a higher incidence of ineffective seizure control, severe retardation and a higher mortality rate. The results suggest that EIMFS patients who present with abnormal MRI, hypsarrhythmia and burst suppression in EEG may be associated with ineffective seizure control and poor prognosis. We need to further expand the sample to analyze and confirm these correlations.

Conclusions
In conclusion, our study expands the EIMFS clinical phenotype and genotype spectra. EIMFS patients who present with abnormal MRI findings, hypsarrhythmia and burst suppression in EEG may be associated with ineffective seizure control and poor prognosis. Etiological analysis showed that in addition to genetic mutations, structural etiology may be associated with poor prognosis. This study is the first to report that ALDH7A1, ATP7A, DOCK6, PRRT2, ALG1, and PCDH19 mutations cause the phenotypic spectrum of EIMFS. The genes KCNT1, SCN1A, ALG1, SCN2A, WWOX and ATP7A may be associated with ineffective seizure control and poor prognosis. Through early diagnosis with genetic tests and the administration of the corresponding precise treatment, the outcomes of EIMFS can be notably improved. According to the clinical characteristics of EIMFS described by Coppola 1 , the inclusion criteria are as follows: (1) onset within 6 months after birth; (2) migrating focal seizures at onset; (3) multifocal seizures intractable to conventional antiepileptic drugs; (4) the EEG during the onset period is characterized by multifocal discharges, migrating in one hemisphere or between the two hemispheres, involving multiple parts, and the clinical onset is closely related to the time and location of the EEG discharge; and (5) delayed developmental progress or signs of psychomotor regression associated with seizure onset. The phenotypic information of all patients was collated on clinical presentation, disease course, EEG, neuroimaging, treatment strategies and the results of neurometabolic and diagnostic genetic investigations. All patients were followed up every 1-6 months by telephone or outpatient department.
It should be noted in particular that one of our patients met the inclusion criteria except for the onset age of 9 months. Considering that EIMFS with onset age of 9 months has been reported in literatures, we still included this patient in our study cohort.
Genetic tests. Genetic testing was carried out using chromosome karyotype analysis, CNV analysis, mitochondrial genome sequencing, epilepsy gene panels and WES. CNV analysis was performed with the Illumina HumanOmniZhonghua-8 Bead Chip; Mitochondrial genome sequencing was subsequently performed on an Illumina HiSeq 2000 platform (Illumina, San Diego, CA, USA); details were provided by our team previously 29 . The epilepsy gene panel contained 265 epilepsy-associated genes was performed on methods previously reported by Lemke et al 30 . WES was also performed based on methods previously reported by our team 29 by using Illumina HiSeq X Ten (Illumina, San Diego, CA, USA) with 150-bp paired-end reads.
Ethics approval and consent to participate. The study was performed in accordance with the Declaration of Helsinki, with the approval of the study protocol by an independent ethics committee or institutional review board at each site. All patients provided written informed consent before participation.

Data availability
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/